Erythropoietin (EPO) is a glycoprotein and a colony stimulating factor which serves as the principal factor involved in the regulation of red blood cell synthesis. Erythropoietin is produced in the kidney and acts by stimulating precursor cells in bone marrow causing them to divide and differentiate into mature red blood cells. Naturally occurring EPO is a glycoprotein containing 165 amino acids that is produced in the kidney. Erythropoietin has been manufactured using recombinant DNA technology through the cloning of the EPO gene and expression in Chinese hamster ovary cells. See Lin, U.S. Pat. No. 5,618,698. The recombinantly produced EPO has been available for some time as an effective therapeutic agent in the treatment of various forms of anemia, including anemia associated with chronic renal failure, zidovidine treated HIV infected patients, and cancer patients on chemotherapy. The glycoprotein is administered parenterally, either as an intravenous (IV) or subcutaneous (SC) injection in conventional buffered aqueous solutions which contain human serum albumin (HSA) as a carrier. Such formulations are marketed in the United States under the trade names EPOGEN® and PROCRIT®. These products contain erythropoietin in 1 ml single dose, preservative-free or 2 ml multidose preserved vials.
While these formulations have been proven to be highly successful, certain disadvantages are associated with the products. Presently, the bioavailability of protein therapeutics such as erythropoietin is limited by short plasma half-lives and the susceptibility to protease degradation. The short half-lives of proteins such as erythropoietin necessitate frequent administration for maximum clinical efficacy. This is disadvantageous for the treatment of chronic conditions and can results in poor patient compliance, reducing efficacy. Accordingly, attempts have been made to increase the plasma half-life of erythropoietin.
In recent years, non-antigenic water-soluble polymers, such as polyethylene glycol (“PEG”) have been used for the covalent modification of polypeptides of therapeutic and diagnostic importance. For example, covalent attachment of PEG to therapeutic polypeptides such as the interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15,064; Tsutsumi, Y. et al., J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al., Drug Des Delivery 1990, 6, 157), catalase (Abuchowski, A. et al., J. Biol Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al., Anal Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al, Biochim, Biophy. Acta 1981, 660, 293), has been reported to extend their half-life in vivo, and/or reduce their immunogenicity and antigenicity.
Derivatized PEG compounds have been previously disclosed (U.S. Pat. No. 5,438,040, Aug. 1, 1995, Conjugation-Stabilized Polypeptide Compositions, Therapeutic Delivery and Diagnostic Formulations Comprising Same, and Method of Making and Using the Same, N. N. Ekwuribe). This approach to post-translational derivatization has also been applied to erythropoietin (EPO). For example, WO 94/28024 discloses carbohydrate modified polymer conjugates with erythropoietin activity wherein the PEG is linked via an oxidized carbohydrate. U.S. Pat. No. 4,904,584 discloses polyalkylene oxide conjugation of lysine-depleted polypeptide variants, including EPO. WO 90/12874 describes the preparation of a monomethoxy-PEG-EPO (mPEG-EPO) in which the EPO contains a cysteine residue introduced by genetic engineering to which the specific PEG reagent is covalently attached. Other PEG-EPO compositions are disclosed in EP 605693, U.S. Pat. No. 6,077,939, WO 01/02017 and EP 539167.
Applicant's co-pending application Ser. No. 09/431,861 discloses the modification of antibodies and antibody fragments with PEG and demonstrates that PEG can increase circulating half-life in mice and primates. Derivatized PEG was used for modification of the Fab fragment of the antibody c7E3. Circulating half-life is increased in direct proportion to the molecular weight of the PEG. As the molecular weight of PEG increases, the activity of the compound to inhibit ADP-induced platelet aggregation in vitro is decreased, while the binding to purified GPIIb/IIIa, as measured by BIAcore, is unaffected. The addition of a fatty acid or a lipid to the PEG (PEG3.4K-DSPE [disteroylphosphatidylethanolamine]) had a greater circulating half-life than did PEG5K. While there is a decrease in the in vitro activity of c7E3 Fab′(PEG5k)2 relative to c7E3 Fab, the activity of c7E3 Fab′-(PEG3.4k-DSPE)2 is equivalent to c7E3 Fab.